1. Field of the Disclosure
The disclosure generally relates to methods of concentrating shear sensitive biopolymers, such as von Willebrand factor (vWF).
2. Brief Description of Related Technology
Known methods of concentrating (and diafiltrating) biopolymers include tangential flow (cross flow) ultrafiltration (and diafiltration) in flat plate and hollow fiber devices. These devices operate at flow rates and transmembrane pressures sufficiently high to ensure a filtrate flux suitable for cost-effective operation. However, these operating conditions create high shear rates. Additionally, these devices may include screens to further increase filtrate flux. These screens also increase the shear stress imparted to biopolymers. Such shear stress is particularly undesirable when attempting to concentrate shear sensitive biopolymers, such as proteins or viral particles, because the stresses can destroy, denature, or inactivate the biopolymer.
There are various known methods to reduce shear stress during concentration and/or diafiltration in flat plate or tangential flow (cross flow) hollow fiber devices. Those methods include reducing the flow rate, increasing the membrane surface area, and increasing the cut-off size of the membrane. However, each of these methods has various problems. For example, reducing the flow rate also reduces the filtrate flux, which undesirably increases the total operation time, increases the risk of membrane fouling, and increases the amount of time the shear sensitive biopolymer is exposed to the shear stress. Increasing the membrane surface area at low flow rates keeps the filtrate flux high and prevents an increase in total operation time. However, at reduced flow rates, the risk of membrane fouling increases. The increased membrane surface area causes more product loss due to increased surface adsorption, costs more for increased membrane area and buffer consumption, and may have a dead volume larger than the desired volume of the product after concentration. Increasing the cut-off size of the membrane results in sufficient filtrate flux due to the larger pore size. However, problems of increased membrane fouling or incompatibility with the shear sensitive biopolymer (i.e., the biopolymer may pass through the membrane and be lost in the filtrate) remain.
Detergents are used in many bio-processing operations to avoid surface adsorption and aggregate formation of proteins. These operations, however, can require specialized buffer additives to stabilize shear sensitive biopolymers.
For tangential flow hollow fiber devices, the recommended shear rate is 2000 to 8000 sec−1 and 2000 to 4000 sec−1 for shear sensitive feed stock. See GE Healthcare, Operating Handbook: Hollow fiber cartridges for membrane separations 8 (2004). However, shear sensitive biopolymers, such as, for example, vWF or viral particles, begin to degrade, denature, or unfold at shear rates above 2000 sec−1. Therefore, there is a need in the art for methods of concentrating shear sensitive biopolymers without imparting high levels of shear stress.
Generally, the prior art does not sufficiently teach or suggest to one of ordinary skill in the art a cost-effective method of concentrating shear sensitive biopolymers without substantial loss of the biopolymer to protein precipitation, membrane fouling, and membrane surface adsorption. Similarly, reducing the flow rate of the biopolymer-containing mixture to thereby reduce the shear stress in devices does not provide an effective alternative because a certain minimum flow rate is needed to avoid membrane fouling and precipitate adsorption.